This project retains its scientific focus on the cellular and molecular biology of four enzymes which catalyze reactions of amino acid and organic acid metabolism and whose inherited deficiency leads to clinically significant metabolic disorders: ornithine transcarbamylase (OTC); propionyl CoA carboxylase (PCC); methylmalonyl CoA mutase (MUT); and cystathionine Beta-synthase (CS). The proposed investigations are aimed specifically at: 1) characterizing further the pathway of biogenesis of the three mitochondrial matrix enzymes in the group (OTC, PCC, MUT) whose subunits are nuclear-encoded, cytoplasmically synthesized, and posttranslationally translocated and processed, with particular emphasis on the systems responsible for mitochondrial recognition, proteolytic cleavage, and assembly into functional enzymes; 2) clarifying the role of posttranslational, limited proteolysis in the physiologic regulation of the only cytosolic enzyme of the group (CS); 3) defining, using cloned cDNAs for PCC, MUT, and CS, the nucleotide sequence of their coding and flanking regions, their respective endonuclease maps, and their genomic organization, and predicting from derived complete amino acid sequence of these polypeptides, the structure of their leader peptides, protease cleavage sites, and cofactor binding sites; 4) testing the thesis that inherited or acquired deficiency of these enzymes in man sometimes results from aberrant sorting or processing; and 5) extending prenatal detection of inherited deficiency of these enzymes to the molecular level using nuclei acid probes and Southern blotting procedures and DNA obtained from biopsies of chorionic villi. Numerous experimental approaches will be employed including: cell-free synthesis of polypeptides programmed with total hepatic RNA or highly enriched individual mRNAs derived therefrom; incubation of mitochondrial protein precursors with intact mitochondria or fractions thereof; isolation from mitochondrial membranes of receptors for mitochondrial protein precursors, and from mitochondrial matrix of the endoprotease(s) which catalyze their cleavage; synthesis of cDNAs followed by their propagation in appropriate plasmid vectors and identification by nuclei acid hybridization or sequence-matching protocols. These studies should add new information concerning both the fundamental regulation of these model enzymatic systems, and their modulation in inherited or acquired disease states.